Prostate cancer, a leading cause of cancer related deaths among men in the United States, is usually diagnosed in the sixth or seventh decades of life, which allows a large window of opportunity for intervention to prevent or slow progression of the disease. Thus, clinical development of agents from these natural products that are relatively safe but can delay onset and/or progression of human prostate cancer is highly desirable. Gugulipid (GL), extract of Commiphora mukul, has been safely used for thousands of years in the Indian Ayurvedic medicine to treat different ailments. GL has been used in many clinical trials that have focused on its cholesterol-lowering effect and the products of standardized formulations of Commiphora mukul are already in human use in U.S. as cholesterol-lowering agents. Supported by R21 CA143104, we, for the first time, have investigated the antitumor activity of GL (a US patent product) in prostate cancer in vitro and in vivo. GL treatment was found to decrease viability of human prostate cancer LNCaP (androgen-dependent) and its androgen-independent variant C81 cells by causing apoptosis induction at pharmacologically achievable concentrations (~1 5mol/L). Interestingly, a normal human prostate epithelial cell line (PrEC) is significantly more resistant to growth inhibition and apoptosis induction by GL, which is a highly desirable feature of potential cancer preventive agents. Most importantly, our preliminary studies showed that oral gavage of GL, thrice per week beginning at five weeks of age for 20 weeks, significantly inhibits prostate cancer incidence and progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice without causing weight loss or any other side effects. Our preliminary studies have also shown that the GL-induced cell death in prostate cancer cells was correlated with generation of reactive oxygen species (ROS) and activation of c-Jun NH2-terminal kinase (JNK). Objective/Hypothesis: we hypothesize that GL-selective killing of prostate cancer cells might target the ROS-mediated mechanism. This R01application proposes preclinical studies to determine the efficacy of GL, a Complementary and Alternative Medicine, for prevention of prostate cancer in vivo and to elucidate the mechanism of its anti-carcinogenic effect. The proposed studies in specific aims 1-3 will determine the mechanism(s) by which GL causes apoptosis and autophagy in human prostate cancer cells. Moreover, investigations of specific aim 4 will evaluate the in vivo efficacy and mechanisms of GL in prevention of prostate cancer. Specific Aims: To test our hypothesis, we propose the following specific aims: 1. Determine whether the selective killing of cancer cells by GL is through a ROS-mediated mechanism using LNCaP, C81 and PC-3 human prostate cancer cells as well as human normal prostate epithelial cell PrEC as a model. The proposed experiments will test whether the GL-mediated apoptosis in human prostate cancer cells is dependent on ROS; and furthermore, whether the mitochondria-derived ROS serve to initiate such GL-induced cell death. 2. Determine the crosstalk between GL-induced autophagy and apoptosis and its mechanisms using LNCaP, PC-3 and PrEC cells as well as PC-3 xenograft model. The propose studies will determine the effect of autophagy induction by GL, and whether GL-mediated autophagy in human prostate cancer cells involves regulation of mitochondrial ROS. Moreover, the real role of GL-caused autophagy in apoptosis induced by GL will be investigated. 3. Determine whether the simultaneous induction of apoptosis and autophagy by GL is regulated by the ROS-dependent regulation of JNK signaling axis using LNCaP, PC-3 and PrEC as a model. Studies are designed to elucidate the mechanism of simultaneous induction of autophagy and apoptosis by GL. Likewise, how does the GL-induced ROS trigger the cancer cell death? Which is downstream of ROS? 4. Determine in vivo efficacy and mechanism of GL for prevention of prostate carcinogenesis and metastasis in TRAMP mice. We will determine the efficacy of orally administered GL against the incidence, metastasis and burden of PIN and prostate carcinoma using the TRAMP mice model. Furthermore, we will evaluate the associated biomarkers of apoptosis and autophagy induced by GL (specific aims 1-3) to use the tumor tissues from the mice treated with vehicle (control) and GL for assay of apoptosis and levels of apoptosis and autophagy regulating proteins. Innovation and Significance: We trust that our proposal falls under the criteria of innovative research because we, for the first time, propose to probe into the effects and molecular mechanisms for prevention of prostate cancer by GL and to determine efficacy of GL for prevention of prostate carcinogenesis using TRAMP transgenic mice. GL is a promising compound and has significant potential in preventive applications since it has selectively anticancer activity for prostate cancer. GL has the potential for immediate clinical translation because a dietary supplement is already in human use for health benefits in America without any toxicity in humans. The long-term intrinsic value of defining the mechanism by which GL causes apoptosis induction resides in the identification of biomarkers of GL response potentially useful in future clinical trials and in the optimization of GL-based regimens for prevention and therapy of prostate cancer.